Orofacial clefts, specifically cleft lip and cleft palate, are common and costly congenital anomalies whose etiologies remain largely unknown. One of the most promising clues to the causes of orofacial clefts is that women who use vitamins containing folic acid in early pregnancy are at much lower risk for cleft-affected pregnancies. Although the underlying mechanisms by which folic acid contributes to these reduced risks are unknown, the evidence suggests that folate intake prevents clefts by compensating for susceptibilities in folate/one-carbon metabolism. However, clear identification of genetic determinants in this pathway through association studies has proven elusive. The approach we propose to further define the molecular genetic mechanisms behind orofacial clefts is based on two previous observations from our group. First, deep sequencing reveals a substantial number of novel, nonsynonymous variants in folate pathway genes (frequencies <=1%) that adversely affect enzyme function, yet are remediable by folate supplementation. Second, a discovery-sequencing study of all folate pathway genes in the context of spina bifida, an anomaly with many similar attributes to isolated clefts, revealed compelling risk signatures only by analyzing biologically relevant allelic combinations. These data suggested that combinations of alleles, both common and rare, are integrated into metabolic function, which ultimately underlies disease risk. Thus, we hypothesize that genetic susceptibilities in one-carbon metabolism may also be etiological for clefts and that these susceptibilities can be conferred by both low-frequency and common alleles, and possibly by synergy between relevant combinations of pathway variants. To test this hypothesis, we will sequence the coding regions in all folate/homocysteine pathway genes (N=32) from a population of ~375 cleft-affected infants and ~375 controls. We will test enzyme variants, individually and in combinations, for their functional impact and nutritional remediation based on quantitative cell-based assays in the yeast S. cerevisiae, and correlate allele distribution and functional studies with clinical phenotype. In addition, we will evaluate the relevance of murine models of orofacial clefts to human etiologies in two ways. First, we will sequence the coding regions of human orthologs of those mouse genes with convincing contributions to lip/palate closure (N=20) to identify the full spectrum of mutation to test whether such genes/variants play a role in human cleft development. Second, we will investigate the hypothesis, based on experiments in mice, that folate exerts its preventive effect through its control of methyl donor flux and subsequent epigenetic changes. Thus, we will explore global DNA methylation in cleft-affected and control newborns, which could lead to logical extensions at specific genomic loci. These studies will better define the causality of orofacial clefts as well as uncover the remedial mechanism of nutritional supplementation. This research plan capitalizes on an ongoing and successful collaboration that unites a unique combination of expertise for its execution.